Protein targeting and transport across lipid bilayers is a fundamental energy-requiring process in all organisms. Up to approximately half of the proteins in an organism's proteome are inserted into or transported across membranes by protein translocation systems, or translocons. Many distinct types of translocation systems exist that allow large protein molecules to cross membranes without compromising the membranes'role as a permeability barrier to ions, metabolic intermediates, and other macromolecules. In order to further expand our knowledge of the molecular mechanisms that exist to translocate large molecules across membranes, the proposed research will examine the bacterial twin-arginine translocation (Tat) export system. The Tat system transports fully-folded and assembled proteins. The number of proteins transported by the Tat system is highly species dependent, ranging from none to many (>100). The absence of a functional Tat system often leads to growth defects in the host bacterium. Further, the Tat machinery is responsible for the export of numerous bacterial virulence factors of human health significance. In a particularly dramatic example, a functional Tat system is required for the growth of Mycobacterium tuberculosis, the causative agent of tuberculosis. Transport by the Tat system minimally requires three proteins, TatA, TatB and TatC. The dominant hypothesis is that a TatBC complex acts as a receptor, which recognizes the presequence of transport substrates, and a TatA oligomer provides a gated pore through which the cargo protein crosses the membrane bilayer. To further understand the basic mechanistic principles governing transport via the Tat system, we will: (1) probe precursor interactions with the lipid and the translocon, by attaching a fluorescence dye to the presequence;(2) construct a kinetic model of transport using a real-time, fluorescence-based transport assay with 1 s time resolution;(3) investigate the role of cargo size and shape on transport rate and transport efficiency;(4) determine the influence of the TatA to TatBC ratio on Tat transport efficiency, transport rate and cargo size restrictions;and (5) develop a single molecule Tat transport assay. These investigations are expected to substantially increase our understanding of how cargos are recognized by the Tat system, and what types of cargos can be translocated. In addition, they will further elucidate the role of the translocon components and the conformational changes required for transport. This characterization of the basic properties of the Tat translocation system will provide an essential foundation for future work, such as the possibility of developing drugs that target the Tat system, or for the utilization of the Tat system in biotechnological applications, such as th expression of protein therapeutics. PUBLIC HEALTH RELEVANCE: The Tat machinery is responsible for the transport of numerous bacterial virulence factors of human health significance, and the absence of a functional Tat system often leads to growth defects in the host bacterium. Consequently, a better understanding of the Tat system is expected to help lead to the development of new antimicrobial drugs. Further, the ability of the Tat system to transport fully-folded protein complexes suggests that it will eventually find utility in the expression of protein therapeutics.